Date Published: May 15, 2014
Publisher: Public Library of Science
Author(s): Sam Alsford, Rachel B. Currier, José Afonso Guerra-Assunção, Taane G. Clark, David Horn, Stephen M. Beverley.
Closely related African trypanosomes cause lethal diseases but display distinct host ranges. Specifically, Trypanosoma brucei brucei causes nagana in livestock but fails to infect humans, while Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense cause sleeping sickness in humans. T. b. brucei fails to infect humans because it is sensitive to innate immune complexes found in normal human serum known as trypanolytic factor (TLF) 1 and 2; the lytic component is apolipoprotein-L1 in both TLFs. TLF resistance mechanisms of T. b. gambiense and T. b. rhodesiense are now known to arise through either gain or loss-of-function, but our understanding of factors that render T. b. brucei susceptible to lysis by human serum remains incomplete. We conducted a genome-scale RNA interference (RNAi) library screen for reduced sensitivity to human serum. Among only four high-confidence ‘hits’ were all three genes previously shown to sensitize T. b. brucei to human serum, the haptoglobin-haemoglobin receptor (HpHbR), inhibitor of cysteine peptidase (ICP) and the lysosomal protein, p67, thereby demonstrating the pivotal roles these factors play. The fourth gene identified encodes a predicted protein with eleven trans-membrane domains. Using chemical and genetic approaches, we show that ICP sensitizes T. b. brucei to human serum by modulating the essential cathepsin, CATL, a lysosomal cysteine peptidase. A second cathepsin, CATB, likely to be dispensable for growth in in vitro culture, has little or no impact on human-serum sensitivity. Our findings reveal major and novel determinants of human-serum sensitivity in T. b. brucei. They also shed light on the lysosomal protein-protein interactions that render T. b. brucei exquisitely sensitive to lytic factors in human serum, and indicate that CATL, an important potential drug target, has the capacity to resist these factors.
The African trypanosomes are flagellated protozoan parasites comprising several species of the genus Trypanosoma, which cause devastating diseases in humans and livestock. One key feature that distinguishes members of this group is their sensitivity to innate trypanolytic factors (TLFs) found in human serum. T. b. brucei and related species cause nagana in livestock but these parasites are rapidly lysed by human TLFs , . T. b. gambiense and T. b. rhodesiense, on the other hand, although sharing >99% genome sequence identity with T. b. brucei, have evolved distinct mechanisms to escape lysis by human serum; these are the causative agents of human African trypanosomiasis (HAT), also known as sleeping sickness, in Western and Eastern Africa, respectively. T. b. gambiense is responsible for 97% of reported cases of HAT .
T. b. gambiense and T. b. rhodesiense can resist the APOL1-based trypanolytic factors found in normal human serum, while T. b. brucei fails to do so. We report here an RNAi library screen in bloodstream-form T. b. brucei for resistance to human serum and identify all three known genes, as well as a novel gene, that increase T. b. brucei susceptibility to this innate immune defence mechanism. We go on to show that one of these genes, encoding inhibitor of cysteine peptidase, acts by modulating the essential activity of CATL, a lysosomal cysteine peptidase. These findings illuminate the interactions between ICP, CATL and human serum, and have important implications for human infectivity, as well as for therapies based on cathepsin inhibitors  or serum lytic factors . Finally, we have revealed a novel role for a putative trans-membrane domain protein, Tb927.8.5240, in determining sensitivity to NHS.